Systemic Sclerosis Fibroblasts Research Articles

Sildenafil Reduces Expression and Release of IL-6 and IL-8 Induced by Reactive Oxygen Species in Systemic Sclerosis Fibroblasts.

Oxidative stress linked to vascular damage plays an important role in the pathogenesis of systemic sclerosis (SSc). Indeed, vascular damage at nailfold capillaroscopy in patients with Raynaud’s Phenomenon (RP) is a major risk factor for the development of SSc together with the presence of specific autoantiobodies. Here, we investigated the effects of the phosphodiesterase type 5 inhibitor (PDE5i) sildenafil, currently used in the management of RP, in modulating the proinflammatory response of dermal fibroblasts to oxidative stress in vitro. Human fibroblasts isolated from SSc patients and healthy controls were exposed to exogenous reactive oxygen species (ROS) (100 µM H2O2), in the presence or absence of sildenafil (1 µM). Treatment with sildenafil significantly reduced dermal fibroblast gene expression and cellular release of IL-6, known to play a central role in the pathogenesis of tissue damage in SSc and IL-8, directly induced by ROS. This reduction was associated with suppression of STAT3-, ERK-, NF-κB-, and PKB/AKT-dependent pathways. Our findings support the notion that the employment of PDE5i in the management of RP may be explored for its efficacy in modulating the oxidative stress-induced proinflammatory activation of dermal fibroblasts in vivo and may ultimately aid in the prevention of tissue damage caused by SSc.

O30 Long non-coding RNA HOTAIR induces beta catenin expression in systemic sclerosis through EZH2 dependent repression of Axin-2

Abstract Background Fibroblasts explanted from affected tissues in systemic sclerosis (SSc) maintain their pro-fibrotic phenotype in vitro. This includes increased secretion of collagens and other extracellular matrix proteins and increased proportion of α-Smooth Muscle Actin (α-SMA) positive cells (myofibroblasts). It has been previously shown that among their profibrotic features, myofibroblasts display activation of WNT signalling, which is linked to a decreased basal expression of AXIN2. Here we aimed to determine whether specific long non-coding RNA (lncRNAs) expressed in myofibroblasts could drive the epigenetically stable decreased expression of Axin 2 in vitro. Methods Full thickness skin biopsies were surgically obtained from the forearms of twelve adult patients with SSc of recent onset. Fibroblasts were isolated and cultured in monolayers and protein and RNA extracted from the fibroblast cultures. Laser capture was performed to isolate cells expressing or not α-SMA as a marker of myofibroblast differentiation. LncRNA HOTAIR was overexpressed in healthy dermal fibroblasts by lentiviral induction. EZH2 was blocked in cultured fibroblasts with the specific inhibitor GSK126. Results HOTAIR expression was increased in SSc patients’ skin (100 fold) and in SSc explanted fibroblasts (5 fold; p < 0.001 for both). Further, laser captured α-SMA expressing fibroblasts expressed in average 2.5 fold higher HOTAIR RNA levels compared to α -SMA negative cells from the same donors (P < 0.05). In vitro, lentiviral induced stable overexpression of HOTAIR in healthy dermal fibroblasts led to their profibrotic activation, including significantly increased expression of Col1A1 and α-SMA both at mRNA and protein levels (2.8 and 1.8 fold respectively, p < 0.05). We further showed that HOTAIR-induced profibrotic activation was due to EZH2 dependent spread of H3k27me3 methylation marker, as demonstrated by complete inhibition by treatment with GSK126. HOTAIR driven EZH2 histone methylation suppressed the expression of Axin 2 in SSc fibroblasts. The reduced Axin 2 levels led to stabilisation of beta catenin and WNT signalling pathway activation. Conclusion Here we show that the epigenetically stable activation of SSc dermal fibroblasts is due to HOTAIR. We also identified a major downstream target of HOTAIR is Axin-2. The results of these studies identify a new venue to modulate fibroblasts biology which could inform clinical research to resolve chronic fibrosis and re-establish tissue homeostasis in SSc. Disclosures C.W. Wasson None. G. Abignano None. R. Ross None. F. Del Galdo Consultancies; AstraZeneca, GSK, Boehringer-Ingelheim, Actelion, Capella Biosciences, Chemomab.

MicroRNA-145 mediates xylosyltransferase-I induction in myofibroblasts via suppression of transcription factor KLF4

Remodelling of the extracellular matrix by myofibroblasts is crucial for wound repair, but if deregulated, it might contribute to the development of fibrosis. Fibroblast-to-myofibroblast differentiation is promoted by aberrant microRNA-145-5p (miR-145) expression in response to transforming growth factor β1 (TGFβ1). One of several myofibroblast markers is human xylosyltransferase-I (XT-I), which is the initial and rate-limiting enzyme of proteoglycan biosynthesis. Increased serum XT activity was quantified in patients with systemic sclerosis (SSc), but the underlying cellular mechanism of this disease remains unknown. This study aims to determine the underlying molecular basis of XT-I induction by considering the miR-mediated regulation of XT-I. We found that miR-145 is upregulated in TGFβ1-treated dermal fibroblasts and correlates with an increased cellular XYLT1 expression and XT activity. Overexpression of miR-145 in dermal fibroblasts induced XYLT1 expression and XT activity and enhanced TGFβ1-promoted XT activity increase. Since direct XYLT1 3′-UTR targeting by miR-145 could be experimentally excluded, an indirect effect of miR-145 on XT-I regulation was indicated. We identified six transcription factor-binding sites for Krueppel-like factor 4 (KLF4), a zinc-finger transcription regulator and putative miR-145 target, in the XYLT1 promoter in silico. A suppressive role of KLF4 on XYLT1 expression was confirmed by targeted gene silencing in dermal fibroblasts and the quantification of KLF4 expression in SSc fibroblasts. Taken together, this study improves the mechanistic understanding of fibrotic remodelling in SSc by identifying a hitherto unknown miR-145/KLF4 pathway mediating the fibrogenic XT-I induction. This knowledge on XYLT1 may lead to the development of novel approaches in the therapy of fibrosis.

015 Inhibitory effect of kaempferol on skin fibrosis in systemic sclerosis by the suppression of oxidative stress

There is growing evidence that vasculopathy-induced hypoxia and oxidative stress enhance the process of fibrosis in systemic sclerosis (SSc). Kaempferol is a natural flavonoid widely found in various vegetables and fruits, and has been reported to have excellent antioxidant activity. Objective was to elucidate the effect of kaempferol on skin fibrosis and the mechanism of the inhibitory regulation of fibrosis by kaempferol. We assessed the effect of intraperitoneally administered kaempferol on bleomycin-induced dermal fibrosis in mice. The effect of kaempferol on oxidative stress in bleomycin-treated mice and SSc fibroblasts was assessed in vivo and in vitro. We identified that kaempferol injection significantly inhibited bleomycin-induced dermal fibrosis in mice. The number of αSMA+ myofibroblasts, CD3+ T-cells, and CD68+ macrophages in lesional skin was significantly decreased by kaempferol injections. Kaempferol administration also significantly suppressed the bleomycin-induced oxidative stress signal in OKD48 mice. Additionally, mRNA levels of oxidative stress-associated factors, such as HO-1 and NOX2, as well as inflammatory and pro-fibrotic cytokines, including IL-6, TGF-β and TNFα in sclerotic skin were significantly decreased by kaempferol. Kaempferol also reduced bleomycin-induced TUNEL+ apoptotic cells in the lesional skin of bleomycin-treated mice. Furthermore, the oxidant-induced intracellular accumulation of reactive oxygen species (ROS) in SSc fibroblasts was inhibited by kaempferol treatment. In addition, the oxidant-induced apoptosis of SSc fibroblasts was decreased by kaempferol in vitro. Kaempferol might improve bleomycin-induced fibrosis by reducing oxidative stress, inflammation, and oxidative cellular damage. Administration of kaempferol might be an alternative treatment for skin fibrosis in SSc.

Interferon regulatory factor 7 (IRF7) represents a link between inflammation and fibrosis in the pathogenesis of systemic sclerosis

ObjectivesThere is considerable evidence that implicates dysregulation of type I interferon signalling (or type I IFN signature) in the pathogenesis of systemic sclerosis (SSc). Interferon regulatory factor 7 (IRF7) has...

Inhibitory effect of kaempferol on skin fibrosis in systemic sclerosis by the suppression of oxidative stress

BackgroundThere is growing evidence that vasculopathy-induced hypoxia and oxidative stress enhance the process of fibrosis in systemic sclerosis (SSc). Kaempferol is a natural flavonoid widely found in various vegetables and fruits, and has been reported to have excellent antioxidant activity. ObjectiveObjective was to elucidate the effect of kaempferol on skin fibrosis and the mechanism of the inhibitory regulation of fibrosis by kaempferol. MethodsWe assessed the effect of intraperitoneally administered kaempferol on bleomycin-induced dermal fibrosis in mice. The effect of kaempferol on oxidative stress in bleomycin-treated mice and SSc fibroblasts was assessed in vivo and in vitro. ResultsWe identified that kaempferol injection significantly inhibited bleomycin-induced dermal fibrosis in mice. The number of αSMA+ myofibroblasts, CD3+ T-cells, and CD68+ macrophages in lesional skin was significantly decreased by kaempferol injections. Kaempferol administration also significantly suppressed the bleomycin-induced oxidative stress signal in OKD48 mice. Additionally, mRNA levels of oxidative stress-associated factors, such as HO-1 and NOX2, as well as inflammatory and pro-fibrotic cytokines, including IL-6, TGF-β and TNFα in sclerotic skin were significantly decreased by kaempferol. Kaempferol also reduced bleomycin-induced TUNEL+ apoptotic cells in the lesional skin of bleomycin-treated mice. Furthermore, the oxidant-induced intracellular accumulation of reactive oxygen species (ROS) in SSc fibroblasts was inhibited by kaempferol treatment. In addition, the oxidant-induced apoptosis of SSc fibroblasts was decreased by kaempferol in vitro. ConclusionKaempferol might improve bleomycin-induced fibrosis by reducing oxidative stress, inflammation, and oxidative cellular damage. Administration of kaempferol might be an alternative treatment for skin fibrosis in SSc.

TGF-β-mediated upregulation of brachyury contributes to constitutively up-regulated type I collagen expression in skin fibroblasts: possible role in systemic sclerosis.

Previous reports have shown that epithelial-to-mesenchymal transition (EMT) indicates the importance of transforming growth factor-β (TGF-β) signalling in the pathogenesis of systemic sclerosis (SSc). However, the underlying molecular mechanisms of EMT are not fully understood. Brachyury, an evolutionarily conserved transcription factor, was recently identified as an important factor that promotes EMT in human carcinoma cell lines. However, there is no evidence indicating that brachyury is involved in EMT in SSc. The expression of brachyury and collagen was investigated in cultures of dermal fibroblasts and skin sections derived from SSc patients and healthy controls. Brachyury and collagen expression were determined by immunohistochemistry and immunoblotting, respectively, and mRNA for both was analysed using real-time PCR. Brachyury was overexpressed in SSc dermal fibroblasts both in vivo and in vitro, and this overexpression was inhibited by TGF-β1 inhibitor. Brachyury siRNA reduced mRNA and protein expression levels of type I collagen in normal and SSc dermal fibroblasts, but did not decrease the levels of major disease-related cytokines. Furthermore, brachyury levels were significantly increased in skin samples of SSc patients relative to healthy controls. The up-regulation of brachyury in response to activated endogenous TGF-β signalling may play a role in constitutive up-regulation of collagen in SSc fibroblasts. Further studies assessing the regulatory mechanism of tissue fibrosis induced by brachyury in SSc skin may lead to a better understanding of the pathogenesis, new diagnostic methods, and new therapeutic approaches using siRNAs.

Downregulation of miR-542-3p Contributes to Apoptosis Resistance in Dermal Fibroblasts from Systemic Sclerosis Patients via Survivin Overexpression

Systemic sclerosis (SSc) is characterized by excessive production of collagens by fibroblasts that leads to vast fibrosis. Resistance to apoptosis is one of the possible underlying mechanisms of fibrosis in these patients. Survivinis involved in inhibition of apoptosis and aberrantly functions in SSc. Since dysregulation of survivin-targeting microRNAs (miRNAs) has frequently been observed in cancer and some autoimmune disorders, this study aimed to investigate their expression status in dermal fibroblasts from SSc patients. DiffuseSSc patients were selected according to American College of Rheumatology criteria. Isolated fibroblasts from 10 SSc and 10 healthy skin biopsies were cultured. After examining purity of the cells, mRNA and miRNAs extraction was performed followed by complementary DNA (cDNA) synthesis. Relative expressions ofsurvivin mRNA, miR-16-5p, miR-320a, miR-218-5p, miR-708-5p and miR-542-3p were analyzed using real time PCR. Survivin mRNA expression was significantly 1.85-fold upregulated in fibroblasts from SSc patients compared with healthy controls (p=0.046). Among the studied miRNAs, miR-542-3p expression was significantly decreased (p=0.033), while enhanced expression of miR-708-5p was observed in SSc fibroblasts (p=0.05) in comparison to healthy subjects. Downregulation of miR-542-3p significantly correlated with survivin overexpression (r=-0.45, p=0.049). Downregulation of miR-542-3p that is correlated with higher surviving expression levels might be a possible cause of apoptosis resistance in SSc fibroblasts, hence providing a new understanding of the disease pathogenesis.

OTUD6B-AS1 Might Be a Novel Regulator of Apoptosis in Systemic Sclerosis.

Antisense long non-coding RNAs (AS lncRNAs) have increasingly been recognized as important regulators of gene expression and they have been found to play key roles in several diseases. However, very little is known about the role of AS lncRNAs in fibrotic diseases such as systemic sclerosis (SSc). Our recent screening experiments by RNA sequencing showed that ovarian tumor domain containing 6B antisense RNA1 (OTUD6B-AS1) and its sense gene OTUD6B were significantly downregulated in SSc skin biopsies. Therefore, we aimed to identify key regulators of OTUD6B-AS1 and to analyze the functional relevance of OTUD6B-AS1 in SSc. OTUD6B-AS1 and OTUD6B expression in SSc and healthy control (HC) dermal fibroblasts (Fb) after stimulation with transforming growth factor-β (TGFβ), Interleukin (IL)-4, IL-13, and platelet-derived growth factor (PDGF) was analyzed by qPCR. To identify the functional role of OTUD6B-AS1, dermal Fb or human pulmonary artery smooth muscle cells (HPASMC) were transfected with a locked nucleic acid antisense oligonucleotide (ASO) targeting OTUD6B-AS1. Proliferation was measured by BrdU and real-time proliferation assay. Apoptosis was measured by Caspase 3/7 assay and Western blot for cleaved caspase 3. While no difference was recorded at the basal level between HC and SSc dermal Fb, the expression of OTUD6B-AS1 and OTUD6B was significantly downregulated in both SSc and HC dermal Fb after PDGF stimulation in a time-dependent manner. Only mild and inconsistent effects were observed with TGFβ, IL-4, and IL-13. OTUD6B-AS1 knockdown in Fb and HPASMC did not affect extracellular matrix or pro-fibrotic/proinflammatory cytokine production. However, OTUD6B-AS1 knockdown significantly increased Cyclin D1 expression at the mRNA and protein level. Moreover, silencing of OTUD6B-AS1 significantly reduced proliferation and suppressed apoptosis in both dermal Fb and HPASMC. OTUD6B-AS1 knockdown did not affect OTUD6B expression at the mRNA level and protein level. Our data suggest that OTUD6B-AS1 regulates proliferation and apoptosis via cyclin D1 expression in a sense gene independent manner. This is the first report investigating the function of OTUD6B-AS1. Our data shed light on a novel apoptosis resistance mechanism in Fb and vascular smooth muscle cells that might be relevant for pathogenesis of SSc.

Bromodomain and Extraterminal (BET) Protein Inhibition Restores Redox Balance and Inhibits Myofibroblast Activation.

Background and Objective Progressive pulmonary fibrosis is the main cause of death in patients with systemic sclerosis (SSc) with interstitial lung disease (ILD) and in those with idiopathic pulmonary fibrosis (IPF). Transforming growth factor-β (TGF-β) and NADPH oxidase- (NOX-) derived reactive oxygen species (ROS) are drivers of lung fibrosis. We aimed to determine the role of the epigenetic readers, bromodomain and extraterminal (BET) proteins in the regulation of redox balance in activated myofibroblasts. Methods In TGF-β-stimulated fibroblasts, we investigated the effect of the BET inhibitor JQ1 on the mRNA expression of the prooxidant gene NOX4 and the antioxidant gene superoxide dismutase (SOD2) by quantitative RT-PCR, the antioxidant transcription factor NF-E2-related factor 2 (Nrf2) activity by a reporter assay, and intracellular ROS levels by dichlorofluorescein staining. Myofibroblast activation was determined by α-smooth muscle actin immunocytochemistry. The role of specific BET protein isoforms in NOX4 gene regulation was studied by siRNA silencing and chromatin-immunoprecipitation. Results and Conclusions Affymetrix gene array analysis revealed increased NOX4 and reduced SOD2 expression in SSc and IPF fibroblasts. SOD2 silencing in non-ILD control fibroblasts induced a profibrotic phenotype. TGF-β increased NOX4 and inhibited SOD2 expression, while increasing ROS production and myofibroblast differentiation. JQ1 reversed the TGF-β-mediated NOX4/SOD2 imbalance and Nrf2 inactivation and attenuated ROS production and myofibroblast differentiation. The BET proteins Brd3 and Brd4 were shown to bind to the NOX4 promoter and drive TGF-β-induced NOX4 expression. Our data indicate a critical role of BET proteins in promoting redox imbalance and pulmonary myofibroblast activation and support BET bromodomain inhibitors as a potential therapy for fibrotic lung disease.

017 Systemic sclerosis fibroblasts show defective activation by coagulation factor XIII in vitro: implications for impaired wound healing in SSc

017 Systemic sclerosis fibroblasts show defective activation by coagulation factor XIII in vitro: implications for impaired wound healing in SSc

A potential new approach for treating systemic sclerosis: Dedifferentiation of SSc fibroblasts and change in the microenvironment by blocking store-operated Ca2+ entry.

Transforming growth factor-β (TGF-β) is an important target for treating systemic sclerosis (SSc). However, our study revealed three levels of TGF-β1 expression in SSc patients, indicating that inhibiting TGF-β is not sufficient to treat SSc. A previous clinical trial also displayed disappointing results. Thus, our study attempted to search for a potential novel approach. Ingenuity Pathway Analysis (IPA) indicated that the SSc pathological pathways were closely associated with store-operated Ca2+ entry (SOCE)-regulated signals, and SOCE activity was found to be increased in SSc fibroblasts. Further treatment of SSc fibroblasts with SOCE inhibitors, 2APB, and associated calcium channel inhibitors SKF96365, and indomethacin, showed that the SOCE inhibitors selectively decreased fibrosis markers and altered the cell morphology. Consequently, SOCE inhibitors, especially 2APB and indomethacin, caused the dedifferentiation of SSc fibroblasts via cytoskeleton remodeling and altered collagen secretion and restored the cell mobility. We further explained SSc pathogenesis as fibroblast differentiation with SOCE. Treatment with exogenous factors, gelatin-1, FAM20A and human albumin, which were identified from the conditioned medium of SSc fibroblasts, was important for regulating the differentiation of fibroblasts with higher levels of SOCE and α-SMA. Conclusively, to treat SSc, blockage of the increased SOCE activity in SSc induces the dedifferentiation of SSc fibroblasts and simultaneously changes the extracellular matrix (ECM) structure to limit SSc pathogenesis.

Epidermal Growth Factor Like-domain 7 and miR-126 are abnormally expressed in diffuse Systemic Sclerosis fibroblasts

Systemic sclerosis (SSc) is characterized by microangiopathy with impaired reparative angiogenesis and fibrosis. Epidermal Growth Factor Like-domain 7 (EGFL7), firstly described in endothelial cells plays a pivotal role in angiogenesis. Fibroblasts (FBs) are involved in vascular remodeling, under physiological and pathological conditions. In this study, we investigated: (i) the expression of EGFL7 and its miR-126 in patients affected by diffuse cutaneous SSc (dcSSc); (ii) the ability of Transforming Growth Factor-beta (TGF-β) to modulate EGFL7 expression; (iii) the ability of EGFL7 to modulate COL1A1 expression and proliferation/migration, and (iv) the functional role of EGFL7 on angiogenesis. Patients were divided in 2 subsets: patients fulfilling the classification criteria in less than one year from Raynaud’s Phenomenon onset (Early Onset Subset–EOS), and all the others (Long Standing Subset–LSS). We show that EGFL7 expression is increased in EOS dcSSc skin and cultured FBs. EGFL7 is inducible by TGF-β on Healthy Controls (HC) FBs but not in SSc-FBs. EGFL7 decreases COL1A1 expression in EOS SSc-FBs while EGFL7 silencing up-regulates COL1A1 expression. EGFL7 promotes migration/invasion of EOS SSc-FBs but not proliferation. Finally, SSc-FBs, partially inhibit angiogenesis in organotypic coculture assays, and this is reversed by treatment with human recombinant (rh)EGFL7. We conclude that EGFL7 and its specific microRNA miR-126 may be involved in the pathogenesis of SSc vasculopathy and fibrosis.

NADPH oxidase-mediated induction of reactive oxygen species and extracellular matrix deposition by insulin-like growth factor binding protein-5.

Insulin-like growth factor binding protein-5 (IGFBP-5) induces production of the extracellular matrix (ECM) components collagen and fibronectin both in vitro and in vivo and is overexpressed in patients with fibrosing lung diseases, such as idiopathic pulmonary fibrosis (IPF) and systemic sclerosis (SSc). However, the mechanism by which IGFBP-5 exerts its fibrotic effect is incompletely understood. Recent reports have shown a substantial role of reactive oxygen species (ROS) in fibrosis; thus we hypothesized that IGFBP-5 induces production of ROS to mediate the profibrotic process. In vitro analyses revealed that ROS production was induced by recombinant and adenoviral vector-mediated IGFBP-5 (AdBP5) in a dose- and time-dependent manner, regulated through MEK/ERK and JNK signaling, and primarily mediated by NADPH oxidase (Nox). Silencing IGFBP-5 in SSc and IPF fibroblasts reduced ROS production. The antioxidants diphenyleneiodonium and N-acetylcysteine blocked IGFBP-5-stimulated ECM production in normal, SSc, and IPF human primary lung fibroblasts. In murine fibroblasts lacking critical components of the Nox machinery, AdBP5-stimulated ROS production and fibronectin expression were reduced compared with wild-type fibroblasts. IGFBP-5 stimulated transcriptional expression of Nox3 in human fibroblasts while selective knockdown of Nox3 reduced ROS production by IGFBP-5. Thus IGFBP-5 mediates fibrosis through production of ROS in a Nox-dependent manner.

Overexpression of apoptosis-related protein, survivin, in fibroblasts from patients with systemic sclerosis.

Recent studies suggest that, in addition to activation and hypersecretion of matrix components, fibroblasts from patients with systemic sclerosis (SSc) are resistant to apoptosis. Previous studies have shown that survivin, a member of inhibition of apoptosis (IAP) family, plays an important role in apoptosis resistance. Accordingly, we decided to study the expression of the most important members of IAP family in SSc fibroblasts, which can block apoptosis either by binding and inhibiting caspases or through caspase-independent mechanisms. Skin biopsy samples were obtained from 19 patients with diffuse cutaneous SSc (DcSSc) and 16 healthy controls. Dermal fibroblasts were cultured and the total RNA was isolated from cells followed by cDNA synthesis. Real-time PCR was performed using SYBR Green PCR master mix and specific primers for cIAP1, cIAP2, XIAP, and Survivin mRNA quantification. A significantly increased expression level of Survivin was observed in fibroblasts from SSc patients compared to controls (2.26-fold, P= 0.04). However, mRNA expression of cIAP1, cIAP2, and XIAP did not change significantly between cases and controls. Our results showed that survivin is upregulated in SSc skin fibroblast which may lead to resistance to apoptosis. Further studies should be performed to reveal the role of survivin in apoptosis pathway of SSc fibroblasts.

Suppressive Regulation by MFG-E8 of Latent Transforming Growth Factor β-Induced Fibrosis via Binding to αv Integrin: Significance in the Pathogenesis of Fibrosis in Systemic Sclerosis.

Several studies have demonstrated that the secreted glycoprotein and integrin ligand milk fat globule-associated protein with epidermal growth factor- and factor VIII-like domains (MFG-E8) negatively regulates fibrosis in the liver, lungs, and respiratory tract. However, the mechanisms and roles of MFG-E8 in skin fibrosis in systemic sclerosis (SSc) have not been characterized. We undertook this study to elucidate the role of MFG-E8 in skin fibrosis in SSc. We assessed expression of MFG-E8 in the skin and serum in SSc patients. We examined the effect of recombinant MFG-E8 (rMFG-E8) on latent transforming growth factor β (TGFβ)-induced gene/protein expression in SSc fibroblasts. We examined the effects of deficiency or administration of MFG-E8 on fibrosis mouse models. We demonstrated that MFG-E8 expression around dermal blood vessels and the serum MFG-E8 level in SSc patients (n = 7 and n = 44, respectively) were lower than those in healthy individuals (n = 6 and n = 28, respectively). Treatment with rMFG-E8 significantly inhibited latent TGFβ-induced expression of type I collagen, α-smooth muscle actin, and CCN2 in SSc fibroblasts (n = 3-8), which suggested that MFG-E8 inhibited activation of latent TGFβ as well as TGFβ signaling via binding to αv integrin. In a mouse model of bleomycin-induced fibrosis (n = 5-8) and in a TSK mouse model (a genetic model of SSc) (n = 5-10), deficient expression of MFG-E8 significantly enhanced both pulmonary and skin fibrosis, and administration of rMFG-E8 significantly inhibited bleomycin-induced dermal fibrosis. These results suggest that vasculopathy-induced dysfunction of pericytes and endothelial cells, the main cells secreting MFG-E8, may be associated with the decreased expression of MFG-E8 in SSc and that the deficient inhibitory regulation of latent TGFβ-induced skin fibrosis by MFG-E8 may be involved in the pathogenesis of SSc and may be a therapeutic target for fibrosis in SSc patients.

Overexpression of ubiquitin-specific peptidase 15 in systemic sclerosis fibroblasts increases response to transforming growth factor β.

ObjectiveUbiquitination of proteins leads to their degradation by the proteasome, and is regulated by ubiquitin ligases and substrate-specific ubiquitin-specific peptidases (USPs). The ubiquitination process also plays important roles in the regulation of cell metabolism and cell cycle. Here, we found that the expression of several USPs is increased in SSc tenosynovial and skin biopsies, and we demonstrated that USP inhibition decreases TGF-β signalling in primary fibroblast cell lines.MethodsHigh-density transcriptomic studies were performed using total RNA obtained from SSc tenosynovial samples. Confirmatory immunostaining experiments were performed on tenosynovial and skin samples. In vitro experiments were conducted in order to study the influence of USP modulation on responses to TGF-β stimulation.ResultsTenosynovial biopsies from SSc patients overexpressed known disease-associated gene pathways: fibrosis, cytokines and chemokines, and Wnt/TGF-β signalling, but also several USPs. Immunohistochemistry experiments confirmed the detection of USPs in the same samples, and in SSc skin biopsies. Exposure of primary fibroblast cell lines to TGF-β induced USP gene expression. The use of a pan-USP inhibitor decreased SMAD3 phosphorylation, and expression of COL1A1, COL3A1 and fibronectin gene expression in TGF-β-stimulated fibroblasts. The effect of the USP inhibitor resulted in increased SMAD3 ubiquitination, and was blocked by a proteasome inhibitor, thereby confirming the specificity of its action.ConclusionOverexpression of several USPs, including USP15, amplifies fibrotic responses induced by TGF-β, and is a potential therapeutic target in SSc.

Research progress in epigenetic studies on systemic sclerosis

Systemic sclerosis (SSc) is an autoimmune disease with unknown etiology, characterized by vasculopathy, inflammation, and extensive fibrosis in the skin and organs. Fibrosis is the hallmark of SSc and contributes to its high mortality. In recent years, with the in-depth study of the epigenetics of SSc (DNA methylation, histone modification, and non-coding RNA), the DNA methylation and miRNA has been the most widely studied. Abnormal DNA methylation can influence the function of vascular endothelial cells, CD4+ T cells, and fibroblasts in SSc. MiRNAs in serum is closely related to autoantibodies, SSc disease activity and complications, and miRNAs in fibroblasts can directly affect the activation of fibroblasts.

Attenuation of aquaporin-3 and epidermal growth factor receptor expression and activation in systemic sclerosis dermal fibroblasts.

Impaired wound healing and skin dehydration are the mainstay of systemic sclerosis (SSc) cutaneous manifestations. Aquaporin-3 (AQP3) has a pivotal role in skin hydration and wound healing. Epidermal growth factor receptor (EGFR) activation is impaired in SSc fibroblasts. It is unclear whether AQP3 downregulation or epidermal growth factor (EGF) signaling are the primary points of dysregulation in SSc patients. Skin punch biopsies were obtained from 10 SSc patients and 10 healthy subjects. The mRNA and/or protein expression levels of AQP3, EGFR/p-EGFR, matrix metalloproteinase-1/2/9 (MMP-1/2/9), and tissue inhibitors of metalloproteinase-1 (TIMP1) at baseline and after EGF and transforming growth factor-β1 (TGF-β1) treatment was evaluated in extracted fibroblasts using real-time polymerase chain reaction and western blot analysis. SSc fibroblasts expressed lower AQP3 and EGFR, compared with normal fibroblasts. Normal fibroblasts increased AQP3 expression in response to EGF whereas AQP3 expression had no change in EGF-treated-SSc fibroblasts. Likewise, EGFR was activated in response to EGF in the normal group but not SSc group. Baseline expression of MMP-1/2/9 and TIMP1 was not different between SSc and controls. EGF treatment did not result in alteration of any MMPs expression in either of the groups. Combination treatment resulted in a significant upregulation of MMP-1 in normal fibroblasts compared with SSc fibroblasts, while in SSc fibroblasts MMP-9 expression was upregulated in response to treatment with TGF-β1 only. Downregulation of AQP3 expression in SSc fibroblasts may be related to reduced EGFR expression and activation. TGF-β1 (alone or in combination with EGF) only can upregulate AQP3 expression in SSc fibroblasts so, TGF-β1 affect MMP-1 and MMP-9 just in SSc fibroblasts.

Systems Analysis of Transcriptomic and Proteomic Profiles Identifies Novel Regulation of Fibrotic Programs by miRNAs in Pulmonary Fibrosis Fibroblasts.

Fibroblasts/myofibroblasts are the key effector cells responsible for excessive extracellular matrix (ECM) deposition and fibrosis progression in both idiopathic pulmonary fibrosis (IPF) and systemic sclerosis (SSc) patient lungs, thus it is critical to understand the transcriptomic and proteomic programs underlying their fibrogenic activity. We conducted the first integrative analysis of the fibrotic programming in these cells at the levels of gene and microRNA (miRNA) expression, as well as deposited ECM protein to gain insights into how fibrotic transcriptional programs culminate in aberrant ECM protein production/deposition. We identified messenger RNA (mRNA), miRNA, and deposited matrisome protein signatures for IPF and SSc fibroblasts obtained from lung transplants using next-generation sequencing and mass spectrometry. SSc and IPF fibroblast transcriptional signatures were remarkably similar, with enrichment of WNT, TGF-β, and ECM genes. miRNA-seq identified differentially regulated miRNAs, including downregulation of miR-29b-3p, miR-138-5p and miR-146b-5p in disease fibroblasts and transfection of their mimics decreased expression of distinct sets of fibrotic signature genes as assessed using a Nanostring fibrosis panel. Finally, proteomic analyses uncovered a distinct “fibrotic” matrisome profile deposited by IPF and SSc fibroblasts compared to controls that highlights the dysregulated ECM production underlying their fibrogenic activities. Our comprehensive analyses of mRNA, miRNA, and matrisome proteomic profiles in IPF and SSc lung fibroblasts revealed robust fibrotic signatures at both the gene and protein expression levels and identified novel fibrogenesis-associated miRNAs whose aberrant downregulation in disease fibroblasts likely contributes to their fibrotic and ECM gene expression.